Process for amplifying nucleic acids

ABSTRACT

The present invention relates to a process for synthesizing or amplifying efficiently a nucleic acid comprising a target nucleic acid sequence. In the process according to the present invention, a primer comprising in its 3′-end portion a sequence (Ac′) which hybridizes a sequence (A) in the 3′-end portion of the target nucleic acid sequence, and in the 5′-side of said sequence (Ac′) a sequence (B′) which hybridizes the complementary sequence (Bc) of a sequence (B) positioned in the 5′-side of said sequence (A) on the target nucleic acid sequence, wherein {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X denotes the number of bases in said sequence (Ac′), Y denotes the number of bases in the region flanked by said sequences (A) and (B) in the target nucleic acid sequence, and Y′ denotes the number of bases in an intervening sequence between said sequences (Ac′) and (B′) (Y′ may be zero).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of application Ser. No. 10/532,975, filedApr. 28, 2005, which is a U.S. National Stage of PCT/JP03/13856, filedOct. 29, 2003, which applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for synthesizing a nucleicacid sequence, which is useful in the field of genetic engineering, aswell as a process for amplifying it. More particularly, the presentinvention relates to a process for synthesizing a nucleic acid sequencewith use of strand displacement reaction, and to a process foramplifying it.

2. Background Art

In the field of genetic engineering, there is known an assay based onthe complementation of nucleic acids as a method which is capable ofdirectly analyzing genetic features. In such assay, if an aimed gene ispresent only in a small amount in a sample, it is necessary topreviously amplify the aimed gene itself generally due to the difficultyof its detection.

The amplification of the aimed gene (amplification of nucleic acid) isprimarily carried out by enzymatic methods with use of DNA polymerase.Such enzymatic methods include, for example, the polymerase chainreaction method (PCR method; U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159), and the reverse transcription PCR method which is thecombination of the PCR method and the reverse transcriptase method(RT-PCR method; Trends in Biotechnology, 10, 146-1521 1992). Thesemethods is intended to make capable of amplifying the aimed gene fromDNA or RNA by repeating the three step reactions of the dissociation(denaturation) of a double-stranded nucleic acid as a template into asingle-stranded nucleic acid, the annealing of a primer to thesingle-stranded nucleic acid and the synthesis (extension) of acomplementary strand from a primer. These methods require the repetitionof three steps in total in which the reaction solution is adjusted to atemperature suitable for each reaction in the three steps describedabove.

There is known the shuttle PCR method as an improvement in theamplification method of nucleic acids described above (“Recent Trends ofthe PCR method”, TANPAKUSHITSU KAKUSAN KOSO (Proteins, Nucleic Acids andEnzymes), KYORITSU SHUPPAN CO., LTD., Vol. 41(5), 425-428 (1996)). Inthe shuttle PCR method, two steps of the annealing of a primer and theextension among the three-step reactions in the PCR method are carriedout at the same temperature, so that the aimed gene can be amplified bythe reactions of two steps in total. Furthermore, EP Laid-OpenPublication No. 0320308 discloses the ligase chain reaction method (LCRmethod), in which a known gene sequence is amplified by two-steptemperature cycling (repeated reactions with heating and cooling).

In the methods described above, it is necessary to use a thermal cyclerwhich can control temperature strictly in an extensive range. Inaddition, these reactions are carried out at two or three temperatureconditions and require time for adjusting respective reactiontemperatures, so that the more increased the cycles, the longer the timerequired for it.

In order to solve the aforementioned problems, methods for amplifyingnucleic acids which can be conducted under an isothermal condition havebeen developed. Such methods include, for example, the stranddisplacement amplification (SDA) method and the self-sustained sequencereplication (3SR) method described in Japanese Patent Publication No.7/114718, the nucleic acid sequence based amplification (NASBA) methodand the transcription-mediated amplification (TMA) method described inJapanese Patent No. 2650159, the Q beta replicase method described inJapanese Patent No. 2710159, a variety of the improved SDA methodsdescribed in U.S. Pat. No. 5,824,517, International PublicationsWO99/09211 or WO95/25180, the LAMP (Loop-Mediated IsothermalAmplification) method described in International Publication WO00/28082,the ICAN (Isothermal and Chimeric primer-initiated Amplification ofNucleic acids) method described in International Publication WO02/16639,and the like. The reactions of all steps involved in the isothermalamplification of nucleic acids proceed simultaneously in reactionmixtures maintained at a constant temperature.

In the SDA method, it is possible to amplify the aimed nucleic acid (andits complementary strand) in a sample by the displacement of a doublestrand mediated by DNA polymerases and restriction endonucleases. Thismethod involves four primers, of which the two primers must be designedto include the recognition sites of the restriction endonucleases. Inaddition, this method requires as a substrate for the synthesis ofnucleic acids modified deoxynucleotide triphosphates such as adeoxynucleotide triphosphate in which the oxygen atom of the phosphategroup at the alpha-position of the triphosphate moiety has beensubstituted by a sulfur atom (S). This method requires high runningcost. Furthermore, in this method, modified nucleotides such asalpha-S-displaced deoxynucleotides are included in the amplified nucleicacid fragment, so that when the amplified fragment is subjected to therestriction enzyme fragment length polymorphism (RFLP) assay, it cannotbe broken with the restriction enzyme and thus such assay cannot bepracticed in some cases.

The SDA method described in U.S. Pat. No. 5,824,517 requires a chimericprimer comprising RNA and DNA, in which DNA is in the 3′-end side. Suchchimeric primer composed of RNA and DNA requires high cost of itssynthesis, and RNA containing primers also require professionalknowledge for its handling. In addition, the improved SDA methoddescribed in International Publication WO99/09211 requires a restrictionenzyme which generates a 5′-protruding end, and the improved SDA methoddescribed in International Publication WO95/25180 requires at least twoprimer pairs, so that these methods also require high running cost.

The ICAN method requires a chimeric primer comprising RNA and DNA, inwhich RNA is in the 3′-end side, and an RNase H for cutting the RNAmoiety at the 3′-end of the primer, so that reagents required for themethod cost a great deal. Thus, this method requires high running costparticularly in genetic tests on a large amount of samples.

The LAMP method requires four primers, which recognize six regions toamplify the aimed genes. That is, in this method, the first primeranneals a template strand to cause extension, and a stem-loop structureis formed at the 5′-end portion of the extended strand due to theconstitution of the first primer. The extended strand is next separatedfrom the template strand by the strand displacement reaction of thesecond primer which is designed in the upper-stream of the first primer.Similar reactions occur repeatedly also on the other strand of thedouble-stranded nucleic acid, and thus the target nucleic acid isamplified. Therefore, the mechanism of the amplification reactions iscomplicated, and the six regions must be selected, so that it becomesdifficult to design primers. In addition, the two of four primers arerequired to be comparatively long chain primers, and thus the synthesisand purification of the primers is expensive and takes a lot of time.

Therefore, there is a need for a process for amplifying nucleic acids,which can be practiced with a low running cost and the nucleic acidfragment thus obtained can be further used for genetic engineeringtreatments. Particularly, it is desired to have an isothermal nucleicacid amplification method in which amplification can be conductedquickly with a pair of primers.

SUMMARY OF THE INVENTION

The present inventors have found that a nucleic acid having a nucleicacid sequence complementary to a target nucleic acid sequence can besynthesized by using a primer designed in such a way that it is capableof forming a stem-loop structure by the extension of the primer andsatisfies additional specific requirements, and that a target nucleicacid can be amplified efficiently by using two such primers. The presentinvention is based on these findings.

Accordingly, it is an object of the present invention to provide aprocess for synthesizing or amplifying efficiently a nucleic acid havinga target nucleic acid sequence, as well as a primer and a primer set foruse therein.

The process for synthesizing a nucleic acid according to the presentinvention is a process for synthesizing a nucleic acid complementary toa target nucleic acid sequence in a template nucleic acid, whichcomprises the steps of:

(a) providing a primer comprising in its 3′-end portion a sequence (Ac′)which hybridizes a sequence (A) in the 3′-end portion of the targetnucleic acid sequence, and in the 5′-side of said sequence (Ac′) asequence (B′) which hybridizes the complementary sequence (Bc) of asequence (B) positioned in the 5′-side of said sequence (A) on thetarget nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Ac′)and (B′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Ac′), and Y denotes the number ofbases in the region flanked by said sequences (A) and (B) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Ac′)and (B′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence;

(b) providing a template nucleic acid;(c) annealing said primer to said template nucleic acid and synthesizinga complementary nucleic acid comprising the complementary sequence ofsaid target nucleic acid sequence by primer extension reaction;(d) hybridizing the sequence (B′) positioned in the 5′-side of thecomplementary nucleic acid synthesized in the step (c) with the sequence(Bc) on the same complementary nucleic acid, thereby allowing theportion of said sequence (A) on the template nucleic acid to besingle-stranded; and(e) annealing another primer having the same sequence as said primer tothe single-stranded sequence (A) portion of the template nucleic acidfrom the step (d) and conducting strand displacement reaction, therebydisplacing the complementary nucleic acid synthesized in the step (c) bythe complementary nucleic acid newly synthesized with said anotherprimer.

The process for amplifying a nucleic acid according to the presentinvention is a process for amplifying a target nucleic acid sequence ina double-stranded template nucleic acid, which comprises the steps of:

(a) providing a first primer comprising in its 3′-end portion a sequence(Ac′) which hybridizes a sequence (A) in the 3′-end portion of thetarget nucleic acid sequence in the first strand of the double-strandedtemplate nucleic acid, and in the 5′-side of said sequence (Ac′) asequence (B′) which hybridizes the complementary sequence (Bc) of asequence (B) positioned in the 5′-side of said sequence (A) on saidtarget nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Ac′)and (B′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Ac′), and Y denotes the number ofbases in the region flanked by said sequences (A) and (B) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Ac′)and (B′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequences;

(b) providing a second primer comprising in its 3′-end portion asequence (Cc′) which hybridizes a sequence (C) in the 3′-end portion ofthe target nucleic acid sequence in the second strand of thedouble-stranded template nucleic acid, and in the 5′-side of saidsequence (Cc′) a sequence (D′) which hybridizes the complementarysequence (Dc) of a sequence (D) positioned in the 5′-side of saidsequence (C) on said target nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Cc′)and (D′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Cc′), and Y denotes the number ofbases in the region flanked by said sequences (C) and (D) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Cc′)and (D′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence;

(c) providing a double-stranded template nucleic acid consisting of thefirst and second template nucleic acids;(d) annealing said first and second primers to said first and secondtemplate nucleic acids, respectively, and synthesizing the first andsecond complementary nucleic acids comprising the complementary sequenceof said target nucleic acid by the primer extension reaction,respectively;(e) hybridizing the sequences (B′) and (D′) positioned in the 5′-side ofthe first and second complementary nucleic acids synthesized in the step(d) with the sequences (Bc) and (Dc) on the same complementary nucleicacids, respectively, and thereby allowing the portions of said sequences(A) and (C) on the first and second template nucleic acids to besingle-stranded, respectively; and(f) annealing another primers having the same sequence as said primersto the single-stranded sequence (A) and (C) portions of the first andsecond template nucleic acids from the step (e) and conducting stranddisplacement reaction, thereby displacing the first and secondcomplementary nucleic acids synthesized in the step (d) by thecomplementary nucleic acids newly synthesized with said another primers.

Furthermore, the primer according to the present invention is a primerfor synthesizing a nucleic acid complementary to a target nucleic acidsequence in a template nucleic acid, comprising in its 3′-end portion asequence (Ac′) which hybridizes a sequence (A) in the 3′-end portion ofthe target nucleic acid sequence, and in the 5′-side of said sequence(Ac′) a sequence (B′) which hybridizes the complementary sequence (Bc)of a sequence (B) positioned in the 5′-side of said sequence (A) on thetarget nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Ac′)and (B′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Ac′), and Y denotes the number ofbases in the region flanked by said sequences (A) and (B) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Ac′)and (B′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence.

The primer set according to the present invention is a primer set foramplifying a target nucleic acid sequence in a double-stranded templatenucleic acid, which comprises:

(a) a first primer comprising in its 3′-end portion a sequence (Ac′)which hybridizes a sequence (A) in the 3′-end portion of the targetnucleic acid sequence in the first strand of the double-strandedtemplate nucleic acid, and in the 5′-side of said sequence (Ac′) asequence (B′) which hybridizes the complementary sequence (Bc) of asequence (B) positioned in the 5′-side of said sequence (A) on saidtarget nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Ac′)and (B′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Ac′), and Y denotes the number ofbases in the region flanked by said sequences (A) and (B) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Ac′)and (B′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence; and (b) a second primer comprising in its3′-end portion a sequence (Cc′) which hybridizes a sequence (C) in the3′-end portion of the target nucleic acid sequence in the second strandof the double-stranded template nucleic acid, and in the 5′-side of saidsequence (Cc′) a sequence (D′) which hybridizes the complementarysequence (Dc) of a sequence (D) positioned in the 5′-side of saidsequence (C) on said target nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Cc′)and (D′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Cc′), and Y denotes the number ofbases in the region flanked by said sequences (C) and (D) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Cc′)and (D′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence.

According to the present invention, it is possible to synthesizecontinuously the target DNA under an isothermal condition by using DNAor RNA as a template and an oligonucleotide primer. According to thepresent invention, it is also possible to amplify continuously thetarget DNA under an isothermal condition by using DNA or RNA as atemplate and a pair of oligonucleotide primers. Thus, the processaccording to the present invention requires no special apparatuses suchas a thermal cycler and no time for the setting of temperature, and thusexhibits an excellent effect that amplified products can be obtained ina short time. Moreover, the DNA fragment amplified according to thepresent invention can be treated with restriction enzymes and thus canbe employed in the field of genetic tests such as restriction enzymefragment length polymorphism or detection of mutation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the schematic diagram of the process for amplifying nucleicacids according to the present invention.

FIG. 2 shows the positions of sequences of a primer used for theamplification of a human STS DYS237 gene in the 5′-side and the 3′-side.

FIG. 3 shows the positions of a primer used for the amplification of ansY160 gene in the 5′-side and 3′-side.

FIG. 4 shows the positions of a primer used for the amplification of anM13mp18RT DNA in the 5′-side and 3′-side.

FIG. 5 shows the amplification of a human STS DYS237 gene under avariety of conditions.

FIG. 6 shows the amplification of a human STS DYS237 gene under avariety of conditions.

FIG. 7 shows the amplification of a human STS DYS237 gene under avariety of conditions.

FIG. 8 shows the amplification of an sY160 gene under a variety ofconditions.

FIG. 9 shows the amplification of an M13mp18RT DNA gene under a varietyof conditions.

FIG. 10 shows the amplification of an M13mp18RT DNA gene under a varietyof conditions.

FIG. 11 shows electrophoresis patterns obtained by the treatment of anamplification product from a human STS DYS237 gene with restrictionenzymes.

FIG. 12 shows electrophoresis patterns obtained by the treatment of anamplification product from an sY160 gene with restriction enzymes.

FIG. 13 shows electrophoresis patterns obtained by the treatment of anamplification product from an M13mp18RT DNA with restriction enzymes.

FIG. 14 shows the amplification of a human STS DYS237 gene in thepresence of a variety of melting temperature adjusting agents.

DETAILED DESCRIPTION OF THE INVENTION

The mechanism of the synthesis of a nucleic acid according to thepresent invention is schematically illustrated in FIG. 1. First, thesequence of a target nucleic acid in a nucleic acid as a template isdetermined, and then the sequence (A) of the 3′-end portion and thesequence (B) positioned in the 5′-side of said sequence (A) on thetarget nucleic acid sequence are determined. The primer according to theinvention comprises a sequence (Ac′), and further comprises a sequence(B′) in the 5′-side thereof. The sequence (Ac′) hybridizes the sequence(A). The sequence (B′) hybridizes the complementary sequence (Bc) of thesequence (B).

In this connection, the primer according to the present invention maycomprise an intervening sequence between said sequences (Ac′) and (B′),the intervening sequence per se having no influence on the reaction. Theannealing of the primer to a template nucleic acid will result in astate in which sequence (Ac′) in the primer has hybridized the sequence(A) of the target nucleic acid (FIG. 1( a)). A nucleic acid comprisingthe complementary sequence of the target nucleic acid is synthesized bythe primer extension reaction in this state. Then, the sequence (B′)positioned in the 5′-end side of the nucleic acid thus synthesizedhybridizes the sequence in the same nucleic acid to form a stem-loopstructure in the 5′-end side of the synthesized nucleic acid. As aresult thereof, the sequence (A) on the template nucleic acid becomes asingle strand, to which another primer having the same sequence as theprevious primer hybridizes (FIG. 1( b)). The nucleic acid synthesizedpreviously is then separated from the template nucleic acid at the sametime as the extension reaction from the newly hybridized primer by thestrand displacement reaction (FIG. 1( c)).

In the aforementioned reaction mechanism, the phenomenon of thehybridization of the sequence (B′) to the sequence (Bc) is caused by thepresence of a complementary region on the same strand. Generally, thedissociation of a double-stranded nucleic acid to single strands beginsfrom a comparatively unstable part such as its terminal or the like. Thedouble-stranded nucleic acid produced by the extension reaction with theprimer described above is in an equilibrium state between thedissociation and bonding of a base pair in the terminal moiety atcomparatively high temperature and maintains a double strand as a whole.If a strand complementary to the dissociated terminal moiety in suchsituation is present in the same strand, a stem-loop structure can beformed as a metastable state. While such stem-loop structure is notpresent stably, the same primer immediately binds to the sequence (A) onthe template nucleic acid as the complementary strand moiety which hasbeen exposed by the formation of the structure, and the extensionreaction with a polymerase causes the liberation of the previouslysynthesized strand and the production of a new double-stranded nucleicacid at the same time.

By repeating the reactions described above, it is possible to synthesizea nucleic acid complementary to the sequence of the target nucleic acidin the template nucleic acid in a large amount. It is also possible tosynthesize nucleic acids in the same manner with a complementary strandof the template nucleic acid described above as a template. Thus, it ispossible to amplify the target nucleic acid in the double-strandedtemplate nucleic acid according to the invention.

The process for synthesizing a nucleic acid according to the inventioncomprises the steps of:

(a) providing a primer comprising in its 3′-end portion a sequence (Ac′)which hybridizes a sequence (A) in the 3′-end portion of the targetnucleic acid sequence, and in the 5′-side of said sequence (Ac′) asequence (B′) which hybridizes the complementary sequence (Bc) of asequence (B) positioned in the 5′-side of said sequence (A) on thetarget nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Ac′)and (B′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Ac′), and Y denotes the number ofbases in the region flanked by said sequences (A) and (B) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Ac′)and (B′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence;

(b) providing a template nucleic acid;(c) annealing said primer to said template nucleic acid and synthesizinga complementary nucleic acid comprising the complementary sequence ofsaid target nucleic acid sequence by primer extension reaction;(d) hybridizing the sequence (B′) positioned in the 5′-side of thecomplementary nucleic acid synthesized in the step (c) with the sequence(Bc) on the same complementary nucleic acid, thereby allowing theportion of said sequence (A) on the template nucleic acid to besingle-stranded; and(e) annealing another primer having the same sequence as said primer tothe single-stranded sequence (A) portion of the template nucleic acidfrom the step (d) and conducting strand displacement reaction, therebydisplacing the complementary nucleic acid synthesized in the step (c) bythe complementary nucleic acid newly synthesized with said anotherprimer.

The term “hybridize” herein means that a part of a primer according tothe invention hybridizes a target nucleic acid under a stringentcondition, but not nucleic acids other than the target nucleic acid. Thestringent condition may be determined depending on factors such as themelting temperatures Tm (° C.) of the double strand of the primeraccording to the invention and its complementary strand and the saltconcentrations of hybridization solutions, and the teachings of thereferences, such as, for example, 3. Sambrook, E. F. Frisch, T.Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory(1989) which is incorporated herein by reference. For example, it ispossible to specifically hybridize a primer to a target nucleic acid byhybridization at a little lower temperature than the melting temperatureused. Such primer can be designed with commercially available primerconstruction softwares such as Primer 3 (Whitehead Institute forBiomedical Research). According to the preferred embodiments of theinvention, a primer hybridizing a certain target nucleic acid comprisesthe all or a part of a nucleic acid molecule complementary to the targetnucleic acid.

The primer according to the invention provided in the step (a) describedabove is constructed in such a way that it is capable of annealing to atemplate nucleic acid in the step (C), and providing the hybridizationof the sequences (B) and (Bc) in the step (d) and a single-strandedsequence (A) to which another primer having the same sequence cananneal. The construction of the primers for conducting preferably thesesteps is described in more details in the following.

In order to conduct efficient annealing of a new primer in the step (e)described above, it is necessary to allow the portion of said sequence(A) on the template nucleic acid to be single-stranded by the formationof the stem-loop structure in the step (d) of the complementary nucleicacid synthesized in the step (c). Thus, it is important to determine(X−Y)/X which is the ratio of the difference (X−Y) to X, in which Xdenotes the number of bases in said sequence (Ac′), and Y denotes thenumber of bases in the region flanked by said sequences (A) and (B) inthe target nucleic acid sequence. In this connection, it is notnecessary to allow also a portion which is positioned in the 5′-side ofthe sequence (A) on the template nucleic acid and which does not affectthe hybridization of the primer to be single-stranded. In addition, theefficient annealing of the new primer in the step (e) described aboverequires the efficient formation of the stem-loop structure in the step(d) on the complementary nucleic acid synthesized in the step (c). It isalso important for the efficient formation of the stem-loop structure,that is, the efficient hybridization of the sequence (B′) and thesequence (Bc), to adjust the distance (X+Y) between the sequences (B′)and (Bc). Generally, the optimal temperature for the primer extensionreaction is up to about 72° C., and thus it is difficult to dissociatethe long region of the extended strand at such lower temperature. It isthus believed more preferred for the efficient hybridization of thesequence (B′) to the sequence (Bc) to have lesser number of basesbetween both sequences. On the other hand, it is believed more preferredfor the hybridization of the sequence (B′) to the sequence (Bc) to allowthe portion of the sequence (A) on the template nucleic acid to besingle-stranded, to have more number of bases between both sequences.

From the viewpoint described above, in the absence of an interveningsequence between said sequences (Ac′) and (B′), the primer according tothe invention is designed in such fashion that (X−Y)/X is in the rangeof −1.00 or more, preferably 0.00 or more, more preferably 0.05 or more,and even more preferably 0.10 or more, and in the range of 1.00 or less,preferably 0.75 or less, more preferably 0.50 or less, and even morepreferably 0.25 or less. Moreover, (X+Y) is preferably in the range of15 or more, more preferably 20 or more, even more preferably 30 or more,and preferably in the range of 50 or less, more preferably 48 or less,and even more preferably 42 or less.

Also, in the presence of an intervening sequence between said sequences(Ac′) and (B′), the primer according to the invention is designed insuch fashion that {X−(Y−Y′)}/X is in the range of −1.00 or more,preferably 0.00 or more, more preferably 0.05 or more, even morepreferably 0.10 or more, and in the range of 1.00 or less, preferably0.75 or less, more preferably 0.50 or less, and even more preferably0.25 or less. Moreover, (X+Y+Y′) is preferably in the range of 15 ormore, more preferably 20 or more, even more preferably 30 or more, andpreferably in the range of 100 or less, more preferably 75 or less, andeven more preferably 50 or less.

The primer according to the invention is composed of deoxynucleotidesand/or ribonucleotides, and has a strand length in which base pairbonding with the target nucleic acid can be conducted while requiredspecificity is maintained under the given condition. The primeraccording to the invention has a strand length in the range ofpreferably 15-100 nucleotides, and more preferably 30-60 nucleotides.Also, the sequences (Ac′) and (B′) have the lengths preferably in therange of 5-50 nucleotides, and more preferably 10-30 nucleotides,respectively. If necessary, an intervening sequence having itself noinfluence on the reaction may be inserted between said sequences (Ac′)and (B′).

In the present invention, the term “ribonucleotide” (also referred to asmerely “N”) means a ribonucleotide triphosphate and includes, forexample, ATP, UTP, CTP, GTP, and the like. Moreover, the ribonucleotidesincludes derivatives thereof such as, for example,alpha-thio-ribonucleotides in which the oxygen atom of alpha-position ofthe phosphate group substituted by a sulfur atom.

In addition, the primers according to the invention includeoligonucleotide primers composed of unmodified deoxynucleotides and/ormodified deoxynucleotides, as well as oligonucleotideprimers composed ofunmodified ribonucleotides and/or modified ribonucleotides, chimericoligonucleotide primers containing unmodified deoxynucleotides and/ormodified deoxynucleotides and unmodified ribonucleotides and/or modifiedribonucleotides, and the like.

The primers according to the invention can be synthesized by any methodswhich can be used for the synthesis of oligonucleotides, such as thephosphate triesterification method, the H-phosphonate method, thethiophosphonate method, and the like. The primers according to theinvention can be easily obtained by the synthetic methods such as, forexample, the phosphoamidite method with use of a DNA synthesizer model394 (ABI, Applied Biosystem Inc.).

The DNA polymerases used in the process for synthesizing nucleic acidsaccording to the invention may be those having strand displacementactivities (strand displacement ability), and either of normaltemperature, mesophilic or thermoduric polymerases may be successfullyused. Also, the DNA polymerases may be either one of natural products orvariants having been artificially varied. Furthermore, the DNApolymerases are preferably those having substantially no 5′->3′exonuclease activities. Such DNA polymerases include, for example, avariant of a DNA polymerase derived from thermophilic bacillus bacteriasuch as Bacillus stearothermophilus (referred to hereinafter as B. st)and Bacillus caldotenax (referred to hereinafter as B. ca) of which the5′→3′ exonuclease activity has been deleted, the Klenow fragment of anE. coli DNA polymerase I, and the like. The DNA polymerases used in theprocess for synthesizing nucleic acids according to the inventionfurther include Vent DNA polymerase, Vent (Exo-) DNA polymerase,DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, cD29 phage DNApolymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfu DNApolymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9° Nm DNApolymerase, Therminater DNA polymerase, and the like.

The other reagents which may be used in the process for synthesizingnucleic acids according to the invention include catalysts such asmagnesium chloride, magnesium acetate, magnesium sulfate, and the like;substrates such as dNTP mix, and the like; and buffers such as Tris-HClbuffer, Tricine buffer, phosphate Na buffer, phosphate K buffer, and thelike. In addition, there may be used additives such as dimethylsulfoxide, and betaine (N,N,N-trimethylglycine), acidic materialsdescribed in International Publication WO99/54455, cationic complexes,and the like.

The nucleic acids used as a template in the process for synthesizingnucleic acids according to the invention may be either DNA or RNA. Thesenucleic acids can be isolated from samples derived from organisms suchas blood, tissues, cells as well as animals or plants, or frommicroorganisms which have been separated from foodstuffs, soils,drainage, and the like.

The template nucleic acid can be isolated by optional methods including,for example, dissolution treatment with surface active agents,sonification, shaking agitation with glass beads, and a method with aFrench press. Also, in the presence of endonuclease, it is preferred topurify the nucleic acid isolated. The nucleic acid can be purified bythe methods such as for example phenol extraction, chromatography,ion-exchange, gel electrophoresis, density-dependent centrifugation, andthe like.

More particularly, it is possible to use either of double-strandednucleic acids such as a genomic DNA or a PCR fragment isolated by themethods described above or single-stranded nucleic acids such as a cDNAprepared by reverse transcription from whole RNA or mRNA. Thedouble-stranded nucleic acid can be optimally used by forming a singlestrand by the denaturing of it.

Enzymes used in the reverse transcription reaction are not particularlylimited except that the enzymes have a cDNA synthesizing activity fromRNA as a template, and include reverse transcriptases derived from avariety of sources such as avian myeloblastosis virus derived reversetranscriptase (AMV RTase), Rous related virus-2 reverse transcriptase(RAV-2 RTase), Moloney murine leukemia virus derived reversetranscriptase (MMLV RTase), and the like. In addition, it is possible touse a DNA polymerase having also a reverse transcription activity. It isalso possible to use Thermus bacteria derived DNA polymerase (TthDNApolymerase and the like), Bacillus bacteria derived DNA polymerase, andthe like. Particularly preferred enzymes include, for example,thermophilic Bacillus bacteria derived DNA polymerases such as B. stderived DNA polymerase, and B. ca derived DNA polymerases (Bca DNApolymerases) such as BcaBEST DNA polymerase, Bca (exo-) DNA polymerase,and the like. By way of example, the Bca DNA polymerase requires nomanganese ion in the reaction, and it is possible to synthesize cDNAwhile suppressing the formation of the secondary structure of a templateRNA under high temperature conditions.

Furthermore, in the process for synthesizing nucleic acids according tothe invention, it is possible to conduct the reverse transcriptionreaction from whole RNA or mRNA and the DNA polymerase reaction in thepresence of cDNA as a template with a polymerase such as BcaBEST DNApolymerase, Bca(exo-) DNA polymerase, and the like. Also, the DNApolymerase may be combined with a reverse transcriptase such as MMLVreverse transcriptase, and the like.

In the process for synthesizing nucleic acids according to theinvention, while it is possible to use the template nucleic acid, evenif it is a double-stranded nucleic acid, directly in the reaction, it isalso possible to carry out efficiently the annealing of a primer to thetemplate nucleic acid after it is denatured into a single strand, ifnecessary. Heating to about 95° C. is a preferred denaturation method.The other denaturation methods also include the denaturation of nucleicacids by ascending pH, but in this case it is necessary to descend pH inorder to hybridize the primer to the target nucleic acid.

According to the preferred embodiment of the present invention, thedouble-stranded nucleic acid obtained in the step (e) is used repeatedlyin the step (d). That is to say, the double-stranded nucleic acidobtained in the step (e) has the same structure as those obtained in thestep (c), and thus it is directly used in the step (d). It is thuspossible to produce in a large scale a nucleic acid complementary to thetarget nucleic acid sequence in the template nucleic acid.

One of the features of the process for synthesizing nucleic acidsaccording to the invention is the practicability in an isothermalcondition. Thus, according to the invention, there is provided a processfor synthesizing a nucleic acid complementary to a target nucleic acidsequence in a template nucleic acid, comprising a step of providing asolution for synthesizing nucleic acids which comprises the templatenucleic acid and the primer according to the invention, and a step ofisothermally incubating the nucleic acid synthesizing solution. In thisconnection, the term “isothermally” means that temperature is maintainedat about constant temperature so as the enzyme and the primer to besubstantially functional.

The process for synthesizing nucleic acids according to the inventioncan be carried out by maintaining the temperature in the range in whichthe activity of the enzyme used is maintained. Also, in order to annealthe primer to the target nucleic acid in the process for synthesizingnucleic acids according to the invention, the reaction temperature ispreferably set in the vicinity of the melting temperature (Tm) of theprimer or less, and the level of stringency is preferably set in view ofthe melting temperature (Tm) of the primer. Thus, the temperature ispreferably in the range of about 20- about 75° C., more preferably inthe range of about 35- about 65° C.

In the process for synthesizing nucleic acids according to theinvention, it is possible to amplify the target nucleic acid sequence ina double-stranded nucleic acid by using the double-stranded nucleic acidas a template and a primer set comprising the two primers according tothe invention designed for each of the strands. Thus, according to theinvention, there is provided a process for amplifying a target nucleicacid sequence in a double-stranded template nucleic acid, whichcomprises the steps of:

(a) providing a first primer comprising in its 3′-end portion a sequence(Ac′) which hybridizes a sequence (A) in the 3′-end portion of thetarget nucleic acid sequence in the first strand of the double-strandedtemplate nucleic acid, and in the 5′-side of said sequence (Ac′) asequence (B′) which hybridizes the complementary sequence (Bc) of asequence (B) positioned in the 5′-side of said sequence (A) on saidtarget nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Ac′)and (B′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Ac′), and Y denotes the number ofbases in the region flanked by said sequences (A) and (B) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Ac′)and (B′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence;

(b) providing the second primer comprising in its 3′-end portion asequence (Cc′) which hybridizes a sequence (C) in the 3′-end portion ofthe target nucleic acid sequence in the second strand of thedouble-stranded template nucleic acid, and in the 5′-side of saidsequence (Cc′) a sequence (D′) which hybridizes the complementarysequence (Dc) of a sequence (D) positioned in the 5′-side of saidsequence (C) on said target nucleic acid sequence, wherein

in the absence of an intervening sequence between said sequences (Cc′)and (D′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotesthe number of bases in said sequence (Cc′), and Y denotes the number ofbases in the region flanked by said sequences (C) and (D) on the targetnucleic acid sequence, and

in the presence of an intervening sequence between said sequences (Cc′)and (D′), {X−(Y−Y′)}/X is in the range of −1.00 to 1.00, in which X andY have the same meanings as above, and Y′ denotes the number of bases insaid intervening sequence;

(c) providing a double-stranded template nucleic acid consisting of thefirst and second template nucleic acids;(d) annealing said first and second primers to said first and secondtemplate nucleic acids, respectively, and synthesizing the first andsecond complementary nucleic acids comprising the complementary sequenceof said target nucleic acid by the primer extension reaction,respectively;(e) hybridizing the sequences (B′) and (D′) positioned in the 5′-side ofthe first and second complementary nucleic acids synthesized in the step(d) with the sequences (Bc) and (Dc) on the same complementary nucleicacids, respectively, and thereby allowing the portions of said sequences(A) and (C) on the first and second template nucleic acids to besingle-stranded, respectively; and(f) annealing another primers having the same sequence as said primersto the single-stranded sequence (A) and (C) portions of the first andsecond template nucleic acids from the step (e) and conducting stranddisplacement reaction, thereby displacing the first and secondcomplementary nucleic acids synthesized in the step (d) by thecomplementary nucleic acids newly synthesized with said another primers.

In this connection, the details on the design of the primers, thereaction conditions and the like are the same as described above on theprocess for synthesizing nucleic acids according to the invention.

According to the preferred embodiments of the invention, thedouble-stranded nucleic acid obtained by the step (f) in the process foramplifying nucleic acids according to the invention is used repeatedlyin the step (e). That is to say, the double-stranded nucleic acidobtained by the step (f) has the same structure as those obtained in thestep (d), and thus it is directly used in the step (e).

According to the other preferred embodiments, the first and secondcomplementary nucleic acids obtained as single-stranded nucleic acids bythe step (f) are used repeatedly as the second and first templatenucleic acids, respectively, in the step (d). That is, the firstcomplementary nucleic acid obtained by the step (f) is used as thesecond template nucleic acid in the step (d), and the secondcomplementary nucleic acid obtained by the step (f) is used as the firsttemplate nucleic acid in the step (d).

The process for amplifying nucleic acids according to the invention canbe practiced isothermally in the similar manner to the process forsynthesizing nucleic acids according to the invention. Thus, accordingto the invention, there is provided a process for amplifying a targetnucleic acid sequence in a double-stranded template nucleic acid,comprising a step of providing a solution for amplifying a nucleic acidwhich comprises the double-stranded template nucleic acid and the primerset according to the invention, and a step of isothermally incubatingthe solution for amplifying the nucleic acid. In this connection, theterm “isothermally” means that temperature is maintained at aboutconstant temperature so as the enzyme and the primer to be substantiallyfunctional. The details on the temperature conditions are the same asdescribed above on the process for synthesizing nucleic acids accordingto the invention.

The process for amplifying the nucleic acid according to the inventioncan be carried out optimally as the process for amplifying a nucleicacid which comprises a step of preparing cDNA from RNA even in the caseof its use as a template by using a DNA polymerase having reversetranscriptase activity such as BcaBEST DNA polymerase. The step ofpreparing cDNA from RNA may be conducted independently, and the productmay be used in the process for amplifying a nucleic acid according tothe invention.

In the process for synthesizing a nucleic acid and the process foramplifying a nucleic acid according to the invention, a meltingtemperature adjusting agent can be added to the reaction solution inorder to enhance the synthetic efficiency or amplification efficiency ofthe nucleic acid. The melting temperature (Tm) of the nucleic acid isgenerally determined by the particular nucleotide sequence of a doublestrand forming portion in the nucleic acid. The melting temperature (Tm)can be changed by adding a melting temperature adjusting agent in thereaction solution, and thus the strength of the double strand formationin the nucleic acid can be adjusted at a fixed temperature. Many meltingtemperature adjusting agents frequently employed have an effect oflowering melting temperatures. The melting temperature of double strandforming portion between two nucleic acids can be lowered, and in otherwords, the strength of forming the double strand can be reduced byadding such melting temperature adjusting agent. Thus, the addition ofsuch melting temperature adjusting agent into a reaction solution in theprocess for synthesizing a nucleic acid and the process for amplifying anucleic acid according to the invention can efficiently change thedouble-stranded portion into a single strand in a nucleic acid regionwhich is rich in GC for forming a strong double strand or in a region inwhich a complicated secondary structure is formed. Thus, the completionof the extension reaction with a primer makes easy the hybridization ofthe next primer on the aimed region, and the synthetic efficiency andamplification efficiency of a nucleic acid can be enhanced. The meltingtemperature adjusting agent used in the invention and its concentrationin a reaction solution is appropriately selected by a person skilled inthe art in consideration of the other reaction conditions which affecthybridization such as salt concentration, reaction temperature, and thelike. Thus, the melting temperature adjusting agents include preferably,but not limited to, dimethyl sulfoxide (DMSO), betaine, formamide orglycerol, or any combinations thereof, and more preferably dimethylsulfoxide (DMSO).

Furthermore, it is also possible to add an enzyme stabilizing agent tothe reaction solution in the process for synthesizing nucleic acids andthe process for amplifying nucleic acids according to the invention. Asa result, the enzyme in the reaction mixture is stabilized, and thesynthetic efficiency and amplification efficiency of nucleic acids canbe enhanced. The enzyme stabilizing agents used in the present inventionmay be any one which is known in the art and includes glycerol withoutlimitation thereto.

In addition, it is also possible to add a reagent for enhancing the heatresistance of enzymes such as DNA polymerase or reverse transcriptase tothe reaction solution in the process for synthesizing nucleic acids andthe process for amplifying nucleic acids according to the invention. Asa result, the enzyme in the reaction mixture is stabilized, and thesynthetic efficiency and amplification efficiency of nucleic acids canbe enhanced. Such reagent may be any one which is known in the art andincludes trehalose without limitation thereto.

In the process for synthesizing nucleic acids and the process foramplifying nucleic acids according to the invention, the synthesisreaction or the amplification reaction are repeated until the enzyme isinactivated or one of the reagents such as the primers has beenexhausted.

In the process for synthesizing nucleic acids and the process foramplifying nucleic acids according to the invention, it is also possibleto use a nucleic acid as a template nucleic acid. The term “non-naturalnucleotide” herein means a nucleotide which contains bases other thanthe bases contained in natural nucleotides (adenine, guanine, cytosine,and thymine or uracil) and is incorporated into a nucleic acid sequence,and includes for example xanthosines, diaminopyridines, isoG, isoC(Proc. Natl. Acad. Sci. USA 92, 6329-6333, 1995), and the like. Targetnucleic acids containing non-natural nucleotides are generally amplifiedwith nucleic acid amplifying enzymes having no heat resistance. On theother hand, the process for synthesizing nucleic acids and the processfor amplifying nucleic acids according to the invention can be conductedisothermally for example at about 50° C., so that the nucleic acidamplifying enzymes such as DNA polymerase will not be inactivated sooften as compared with the conventional PCR method. Thus, the processfor synthesizing nucleic acids and the process for amplifying nucleicacids according to the invention can also be effectively employed forthe amplification of non-natural nucleotide-containing target nucleicacids in which the nucleic acid-amplifying enzymes having no heatresistance are used. Enzymes used for the amplification of nucleic acidscontaining non-natural nucleotides may be the ones which are capable ofamplifying such target nucleic acids without any further limitations,and preferably include a Y188L/E478Q mutated HIV I reversetranscriptase, an AMV reverse transcriptase, a Klenow fragment of a DNApolymerase, a 9° N DNA polymerase, a HotPub DNA polymerase, and the like(see Michael Sismour 1 et al., Biochemistry 42(28), 8598, 2003/U.S. Pat.No. 6,617,106; Michael 3. Lutz et al., Bioorganic & Medical ChemistryLetters 8, 1149-1152, 1998; etc.). Moreover, it is also possible to addmaterials for improving the heat resistance of nucleic acid-amplifyingenzymes, such as trehalose, to a reaction solution, and thus non-naturalnucleotide-containing target nucleic acids can be amplified moreefficiently.

It is possible to prepare quickly a single-stranded nucleic acid forimmobilizing on a DNA chip, a single-stranded DNA probe for determiningthe base sequence or a megaprimer for the long chain PCR method by usingthe process for synthesizing nucleic acids or the process for amplifyingnucleic acids according to the invention. For instance, it is possibleto selectively amplify only a sense sequence or an antisense sequenceaccording to objects by using the process for synthesizing nucleic acidsor the process for amplifying nucleic acids according to the invention.Therefore, the process for synthesizing nucleic acids or the process foramplifying nucleic acids according to the invention are also useful asthe process for producing the sense or antisense sequence of a certaintarget nucleic acid.

Amplified products obtained by the process for synthesizing nucleicacids or the process for amplifying nucleic acids according to theinvention can be detected by any appropriate methods. One of the methodsis the detection of an amplified product having a specific size by theconventional gel electrophoresis. According to this method, theamplified product can be detected with fluorescent materials such asethidium bromide, SYBR Green, and the like. In another method, theproduct can also be detected by hybridizing a labeled probe having alabel such as biotin with the product. Biotin can be detected by bindingwith fluorescent-labeled avidin or with avidin bound to an enzyme suchas peroxidase.

Also, the amplified product obtained by the process for synthesizingnucleic acids or the process for amplifying nucleic acids according tothe invention can be detected with an immunochromatograph. In thismethod, it is devised to employ a chromatographic medium with amacroscopically detectable label (immunochromatography technique).Hybridization of the amplified fragment and the labeled probe, andimmobilization of a capturing probe, which is capable of hybridizingwith the other different sequences in the amplified fragment, on thechromatographic medium makes it possible to trap the product at theimmobilized part and to detect it in the chromatographic medium. As aconsequence, macroscopically simple detection of the product can beconducted.

Further, in the process for synthesizing nucleic acids or the processfor amplifying nucleic acids according to the invention, a primerimmobilized on beads can be used for confirming the agglutination of thebeads due to the synthesis or amplification of a nucleic acid and thusdetecting the synthetic or amplification product. Also, in order tosynthesize or amplify a plurality of target nucleic acids, each primerdesigned with regard to each of the target nucleic acids can beimmobilized on beads, which are different in color, shape or the likeand thus can be distinguished from one another, for the reaction ofsynthesizing or amplifying nucleic acids in a reaction solutioninvolving these beads. In such case, whether the respective targetnucleic acids is present or not is recognized by confirming the presenceor absence of the agglutination of respective beads.

Furthermore, in the process for synthesizing nucleic acids or theprocess for amplifying nucleic acids according to the invention, aprimer immobilized on an array such as e.g. DNA chip can be used forconfirming the agglutinated nucleic acids produced on the array due tothe synthesis or amplification of a nucleic acid and thus detecting thesynthetic or amplification product. Also, in order to synthesize oramplify a plurality of target nucleic acids, each primer designed withregard to each of the target nucleic acids can be immobilized on anarray, which can be distinguished from one another, for the reaction ofsynthesizing or amplifying nucleic acids in a reaction solutioninvolving the array. In such case, whether the respective target nucleicacids is present or not is recognized by confirming the presence orabsence of the agglutinated nucleic acid at the corresponding positionson the array. It is also possible to use an intercalater in place of theconfirmation of the agglutinated nucleic acid.

The amplified fragment obtained by the process for amplifying a nucleicacid according to the invention is composed of ordinary bases, and thuscan be subcloned into an appropriate vector by using a restrictionenzyme site within the amplified fragment. In addition, it is alsopossible to carry out treatment with restriction enzymes such as RFLP,which can be employed widely in the field of genetic test as well. Also,since the amplified fragment obtained by the process for amplifying anucleic acid according to the invention is composed of ordinary bases,the incorporation of the promoter sequence of an RNA polymerase into theamplified fragment makes it possible to synthesize an RNA directly fromthe amplified fragment, and the RNA can also be used as an RNA probe.

Furthermore, in the process for synthesizing nucleic acids or theprocess for amplifying nucleic acids according to the invention, a baselabeled with biotin or a fluorescent material can be used in place of anordinary dNTP to prepare a DNA probe labeled with biotin or thefluorescent material.

The single-stranded nucleic acid prepared by the process forsynthesizing nucleic acids or the process for amplifying nucleic acidsaccording to the invention can be used as a DNA fragment immobilized onthe DNA chip. That is to say, the process for synthesizing nucleic addsor the process for amplifying nucleic acids according to the inventioncan be applied also to the method for preparing a DNA strand immobilizedin the preparation of a DNA chip. It is also possible to prepare a DNAchip by preliminarily immobilizing the 5′-end of a primer on the DNAchip, on which the synthesis or amplification of a nucleic acid iscarried out. It is also possible to conduct the real time detectiontogether with the synthesis or amplification of a nucleic acid on theDNA chip by preliminarily adding a fluorescent labeling probe prior tothe synthesis or amplification of a nucleic acid.

In order to practice the process for synthesizing nucleic acids or theprocess for amplifying nucleic acids according to the invention,reagents involved in the process can be combined to make a kit. Thus,the kit according to the invention comprises a primer or a primer setaccording to the invention. Also, the process for synthesizing nucleicacids or the process for amplifying nucleic acids according to theinvention has an advantage that the process requires no primers otherthan the primer or the primer set according to the invention. Thus,according to the preferred embodiments of the invention, the kitaccording to the invention comprises no primer ingredients other thanthe primer or the primer set according to the invention. The kitaccording to the invention may further include reagents, reactionvessels, instructions described above, and the like.

EXAMPLES

The invention is further described more particularly in the followingexamples, which should not be construed in any way as restrictions onthe invention.

Example 1

In this example, the amplification of a human STS DYS237 gene wasattempted with Human DNA (Clontech) as a template. Primers employed wereas follows. These primers were synthesized by the consignment to ESPECOLIGO SERVICE CORP.

The features of the primers used in the experiment were described below.The relationships of respective primers to the template were asillustrated in FIG. 2. In this connection, underlined parts in thefollowing sequences represent 3′-end regions common to each of senseprimers and antisense primers, respectively.

Primer set 1: a combination of primers comprising solely sequencesannealing to the template (20mer);

SY153L: GCATCCTCATTTTATGTCCA; (SEQ ID NO: 1)SY153R: CAACCCAAAAGCACTGAGTA. (SEQ ID NO: 2)

Primer set 2: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (13mer) is hybridizedwith a region starting from a base downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP13-0: AAGTCTCTGATGTGCATCCTCATTTTATGTCCA; (SEQ ID NO: 3)SY153RP13-0: AGAACTCGCTTTACAACCCAAAAGCACTGAGTA. (SEQ ID NO: 4)

Primer set 3: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (13mer) is hybridizedwith a region starting from six bases downstream of the 3′-end portionof the respective primers on a strand extended from the primer;

SY153LP13-5: GTATTAAGTCTCTGCATCCTCATTTTATGTCCA; (SEQ ID NO: 5)SY1535P13-5: CACTAAGAACTCGCAACCCAAAAGCACTGAGTA. (SEQ ID NO: 6)

Primer set 4: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (13mer) is hybridizedwith a region starting from 11 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP13-10: GTTCAGTATTAAGGCATCCTCATTTTATGTCCA; (SEQ ID NO: 7)SY153RP13-10: AGCATCACTAAGACAACCCAAAAGCACTGAGTA. (SEQ ID NO: 8)

Primer set 5: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (13mer) is hybridizedwith a region starting from 16 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP13-15: CATTTGTTCAGTAGCATCCTCATTTTATGTCCA; (SEQ ID NO: 9)SY153RP13-15: CTTGCAGCATCACCAACCCAAAAGCACTGAGTA. (SEQ ID NO: 10)

Primer set 6: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (10mer) is hybridizedwith a region starting from 21 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP10: GGCATTTGTTGCATCCTCATTTTATGTCCA; (SEQ ID NO: 11) SY153RP10:ATCTTGCAGCCAACCCAAAAGCACTGAGTA. (SEQ ID NO: 12)

Primer set 7: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (13mer) is hybridizedwith a region starting from 21 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP13: TGTGGCATTTGTTGCATCCTCATTTTATGTCCA; (SEQ ID NO: 13)SY153RP13:  AACATCTTGCAGCCAACCCAAAAGCACTGAGTA. (SEQ ID NO: 14)

Primer set 8: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (16mer) is hybridizedwith a region starting from 21 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

(SEQ ID NO: 15) SY153LP16: TTATGTGGCATTTGTTGCATCCTCATTTTATGTCCA;(SEQ ID NO: 16) SY153RP16: CTTAACATCTTGCAGCCAACCCAAAAGCACTGAGTA.

Primer set 9: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (22mer) is hybridizedwith a region starting from 21 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP22: (SEQ ID NO: 17) TTACCTTTATGTGGCATTTGTTGCATCCTCATTTTATGTCCA;SY153RP22: (SEQ ID NO: 18) ATTTAACTTAACATCTTGCAGCCAACCCAAAAGCACTGAGTA.

Primer set 10: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (25mer) is hybridizedwith a region starting from 21 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP25: (SEQ ID NO: 19)TCATTACCTTTATGTGGCATTTGTTGCATCCTCATTTTATGTCCA; SY153RP25:(SEQ ID NO: 20) AAGATTTAACTTAACATCTTGCAGCCAACCCAAAAGCACTGAGTA.

Primer set 11: a combination of primers in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template andextension reaction, a sequence in the 5′-end side (28mer) is hybridizedwith a region starting from 21 bases downstream of the 3′-end portion ofthe respective primers on a strand extended from the primer;

SY153LP28: (SEQ ID NO: 21)CAGTCATTACCTTTATGTGGCATTTGTTGCATCCTCATTTTATGTCCA; SY153RP28:(SEQ ID NO: 22) AAGAAGATTTAACTTAACATCTTGCAGCCAACCCAAAAGCACTGAGTA.

A reaction mixture (25 μl) of Tris-HCl (20 mM, pH8.8), KCl (10 mM),(NH₄)₂SO₄ (10 mM), MgSO₄ (2 mM), Triton X-100 (0.1%), dNTP (0.4 mM), aprimer pair (100 μmol, resp.), a template DNA (100 ng), and Bst DNApolymerase (8U; NEW ENGLAND BioLabs) was prepared, and incubated at 60°C. for 20, 40, or 60 minutes.

A 5 μl portion of each mixture was subjected to electrophoresis in 3%NuSieve GTG Agarose (manufactured by BioWhittaker Molecular Applications(BMA); purchased from Takara Bio Inc.; “NuSieve” is the registeredtrademark of BMA). Results are shown in FIGS. 5, 6 and 7. Samples inrespective lanes in these figures are shown in the following Tables 1-3.

TABLE 1 Explanation of lanes of electrophoretic photograms in FIG. 5Lane Primer Template Reaction Time (min.) 1 DNA size marker (20 bpladder) 2 Primer set 1 Yes 20 3 Primer set 1 Yes 40 4 Primer set 1 Yes60 5 Primer set 1 No 60 6 Primer set 2 Yes 20 7 Primer set 2 Yes 40 8Primer set 2 Yes 60 9 Primer set 2 No 60 10 Primer set 3 Yes 20 11Primer set 3 Yes 40 12 Primer set 3 Yes 60 13 Primer set 3 No 60 14Primer set 4 Yes 20 15 Primer set 4 Yes 40 16 Primer set 4 Yes 60 17Primer set 4 No 60 18 Primer set 5 Yes 20 19 Primer set 5 Yes 40 20Primer set 5 Yes 60 21 Primer set 5 No 60

TABLE 2 Explanation of lanes of electrophoretic photograms in FIG. 6Lane Primer Template Reaction Time (min.) 1 DNA size marker (20 bpladder) 2 Primer set 6 Yes 20 3 Primer set 6 Yes 40 4 Primer set 6 Yes60 5 Primer set 6 No 60 6 Primer set 7 Yes 20 7 Primer set 7 Yes 40 8Primer set 7 Yes 60 9 Primer set 7 No 60 10 Primer set 8 Yes 20 11Primer set 8 Yes 40 12 Primer set 8 Yes 60 13 Primer set 8 No 60 14Primer set 9 Yes 20 15 Primer set 9 Yes 40 16 Primer set 9 Yes 60 17Primer set 9 No 60 18 Primer set 10 Yes 20 19 Primer set 10 Yes 40 20Primer set 10 Yes 60 21 Primer set 10 No 60

TABLE 3 Explanation of lanes of electrophoretic photograms in FIG. 7Lane Primer Template Reaction Time (min.) 1 DNA size marker (20 bpladder) 2 Primer set 11 Yes 20 3 Primer set 11 Yes 40 4 Primer set 11Yes 60 5 Primer set 11 No 60

In Lanes 5, 9, 13, 17 and 21 of respective Figures, no bands other thanthose of stained unreacted primers were observed due to the addition ofno template.

In Lanes 2 and 3 of FIG. 5, bands of an unreacted primer and a templatehaving a high molecular size were confirmed because of the addition of atemplate. However, no amplified products were confirmed because ofinsufficient reaction time. As shown in Lane 4 of FIG. 5, in the samplehaving a template added thereto and reacted for 60 minutes were obtainedan amplified product, which was in the form of a ladder in the low sizeregion and of a smear in the high size region. In Lanes 2-5 of FIG. 5,Primer set 1 containing solely an oligonucleotide (20mer) which annealsto a template was used and no synthetic reaction occurred, so that noamplified products as the object were obtained.

In Lane 6 and the subsequent lanes of FIG. 5, there is shown the resultsof amplifications with a primer set in which after annealing ofsequences placed in the 3′-end side of the respective primers (20mer:sequences identical to those in Primer set 1) to the template, asequence in the 5′-end side is hybridized with a region starting frombases downstream of the 3′-end portion of the respective primers on theextended strand of the primers.

As shown in Lanes 8 and 12 of FIG. 5, when Primer sets 2 or 3 were used,it was possible to obtain the aimed amplification product in a reactiontime of 60 minutes. Of the low size bands, the band in the vicinity ofca. 160 by is an expected product of the synthetic reaction of theinvention.

Further, as shown in Lanes 15 and 16 of FIG. 5, Lanes 3, 4, 7, 8, 11,12, 15, 16, 19 and 20 of FIG. 6, and Lanes 3 and 4 of FIG. 7, it waspossible to obtain the aimed amplification product in a reaction time of40 minutes or more when Primer sets 4, 6, 7, 8, 9, 10 and 11 were used.Of the low size bands, the band in the vicinity of ca. 160 bp is anexpected product of the synthetic reaction of the invention.

In addition, as shown in Lanes 18-20 of FIG. 5, it was possible toobtain the aimed amplification product in a reaction time of 20 minutesor more when Primer set 5 was used. Of the low size bands, the band inthe vicinity of ca. 160 by is an expected product of the syntheticreaction of the invention.

When the distance between the region corresponding to the sequence inthe 3′-end side of the primer on the extended strand of the primer andthe region with which the sequence in the 5′-end side hybridizes iscomparatively small as in Primer sets 2 and 3, it is considered that along reaction time is required because most of the sequence on atemplate having the same sequence to which the next primer is to beannealed remains as a double strand and the subsequent annealing hardlyoccurs.

Also, when the distance between the region corresponding to the sequencein the 3′-end side of the primer on the extended strand of the primerand the region with which the sequence in the 5′-end side hybridizes iscomparatively large as in Primer sets 6-11, it is considered that acomparatively long reaction time is required because the foldingefficiency of the sequence in the 5′-end side of each primer is lowered.

On the other hand, when the distance between the region corresponding tothe sequence in the 3′-end side of the primer on the extended strand ofthe primer and the region with which the sequence in the 5′-end sidehybridizes is not excessively small or excessively large as in Primerset 5, it is considered that the most efficient amplification can beperformed in the invention.

Example 2

In this example, the amplification of an sY160 gene was attempted withHuman DNA (Clontech) as a template. Primers employed were as follows.These primers were synthesized by the consignment to ESPEC OLIGO SERVICECORP.

The features of the primers used in the experiment were described below.The relationships of respective primers to the template were asillustrated in FIG. 3. In this connection, underlined parts in thefollowing sequences represent 3′-end regions common to each of senseprimers and antisense primers, respectively.

Primer set 12: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (20mer) to thetemplate and extension reaction, a sequence in the 5′-end side (13mer)is hybridized with a region starting from 27 bases downstream of the3′-end portion of the primer on a strand extended from the primer, andan antisense primer in which after annealing of a sequence placed in the3′-end side of a primer (20mer) to the template and extension reaction,a sequence in the 5′-end side (13mer) is hybridized with a regionstarting from 21 bases downstream of the 3′-end portion of the primer ona strand extended from the primer;

SY160LP13: ATTCGATTCCGTTTACGGGTCTCGAATGGAATA; (SEQ ID NO: 23) SY160RP13:CTAAATCGAATGGTCATTGCATTCCTTTCCATT. (SEQ ID NO: 24)

Primer set 13: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (20mer: sequenceidentical to that in Primer set 12) to the template and extensionreaction, a sequence in the 5′-end side (13mer) is hybridized with aregion starting from 27 bases downstream of the 3′-end portion of theprimer on a strand extended from the primer, and an antisense primer inwhich after annealing of a sequence placed in the 3′-end side of aprimer (20mer: sequence identical to that in Primer set 12) to thetemplate and extension reaction, a sequence in the 5′-end side (16mer)is hybridized with a region starting from 21 bases downstream of the3′-end portion of the primer on a strand extended from the primer;

(SEQ ID NO: 25) SY160LP16: GACATTCGATTCCGTTTACGGGTCTCGAATGGAATA;(SEQ ID NO: 26) SY160RP16: GAACTAAATCGAATGGTCATTGCATTCCTTTCCATT.

A reaction mixture (25 μl) of Tris-HCl (20 mM, pH8.8), KCl (10 mM),(NH₄)₂SO₄ (10 mM), MgSO₄ (2 mM), Triton X-100 (0.1%), dNTP (0.4 mM), aprimer pair (100 μmol, resp.), a template DNA (100 ng), and Bst DNApolymerase (8U; NEW ENGLAND BioLabs) was prepared, and incubated at 60°C. for 60 or 90 minutes.

A 5 μl portion of each mixture was subjected to electrophoresis in 3%NuSieve GTG Agarose (manufactured by BioWhittaker Molecular Applications(BMA); purchased from Takara Bio Inc.; “NuSieve” is the registeredtrademark of BMA). Results are shown in FIG. 8. Samples in respectivelanes in these figures are shown in the following Table 4.

TABLE 4 Explanation of lanes of electrophoretic photograms in FIG. 8Lane Primer Template Reaction Time (min.) 1 DNA size marker (20 bpladder) 2 Primer set 12 Yes 60 3 Primer set 12 Yes 90 4 Primer set 12 No90 5 Primer set 13 Yes 60 6 Primer set 13 Yes 90 7 Primer set 13 No 90

In Lanes 4 and 7, no bands other than those of stained unreacted primerswere observed due to the addition of no template.

In Lanes 2 and 5, bands of an unreacted primer and a template having ahigh molecular size were confirmed because of the addition of atemplate. However, no amplified products were confirmed because ofinsufficient reaction time. As shown in Lane 3 and 6, in the samplehaving a template added thereto and reacted for 90 minutes were obtainedan aimed amplified product in a satisfactory amount. Of the low sizebands, the one in the vicinity of ca. 260 by is an expected product ofthe synthetic reaction of the invention.

Example 3

In this example, the amplification of an M13mp18RF DNA (phage vector;TAKARA BIO INC.) was attempted with the same DNA as a template. Primersemployed were as follows. These primers were synthesized by theconsignment to ESPEC OLIGO SERVICE CORP.

The features of the primers used in the experiment were described below.The relationships of respective primers to the template were asillustrated in FIG. 4. In this connection, underlined parts in thefollowing sequences represent 3′-end regions common to each of senseprimers and antisense primers, respectively.

Primer set 14: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (24mer) to thetemplate and extension reaction, a sequence in the 5′-end side (24mer)is hybridized with a region starting from 51 bases downstream of the3′-end portion of the primer on a strand extended from the primer, andan antisense primer in which after annealing of a sequence placed in the3′-end side of a primer (22mer) to the template and extension reaction,a sequence in the 5′-end side (25mer) is hybridized with a regionstarting from 54 bases downstream of the 3′-end portion of the primer ona strand extended from the primer;

M13BIP: (SEQ ID NO: 27)CGACTCTAGAGGATCCCCGGGTACTGTTGTGTGGAATTGTGAGCGGAT; M13FIP:(SEQ ID NO: 28) ACAACGTCGTGACTGGGAAAACCCTGTGCGGGCCTCTTCGCTATTAC.

Primer set 15: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (24mer: sequenceidentical to that in Primer set 14) to the template and extensionreaction, a sequence in the 5′-end side (13mer) is hybridized with aregion starting from one base downstream of the 3′-end portion of theprimer on a strand extended from the primer, and an antisense primer inwhich after annealing of a sequence placed in the 3′-end side of aprimer (22mer: sequence identical to that in Primer set 14) to thetemplate and extension reaction, a sequence in the 5′-end side (13mer)is hybridized with a region starting from one base downstream of the3′-end portion of the primer on a strand extended from the primer;

(SEQ ID NO: 29) M13F2L13-0: GTGTGAAATTGTTTGTTGTGTGGAATTGTGAGCGGAT;(SEQ ID NO: 30) M13R2L13-0: TTCGCCAGCTGGCGTGCGGGCCTCTTCGCTATTAC.

Primer set 16: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (24mer: sequenceidentical to that in Primer set 14) to the template and extensionreaction, a sequence in the 5′-end side (13mer) is hybridized with aregion starting from seven bases downstream of the 3′-end portion of theprimer on a strand extended from the primer, and an antisense primer inwhich after annealing of a sequence placed in the 3′-end side of aprimer (22mer: sequence identical to that in Primer set 14) to thetemplate and extension reaction, a sequence in the 5′-end side (13mer)is hybridized with a region starting from seven bases downstream of the3′-end portion of the primer on a strand extended from the primer;

(SEQ ID NO: 31) M13F2L13-6: TTTCCTGTGTGAATGTTGTGTGGAATTGTGAGCGGAT;(SEQ ID NO: 32) M13R2L13-6: CCCCCTTTCGCCAGTGCGGGCCTCTTCGCTATTAC.

Primer set 17: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (24mer: sequenceidentical to that in Primer set 14) to the template and extensionreaction, a sequence in the 5′-end side (13mer) is hybridized with aregion starting from 13 bases downstream of the 3′-end portion of theprimer on a strand extended from the primer, and an antisense primer inwhich after annealing of a sequence placed in the 3′-end side of aprimer (22mer: sequence identical to that in Primer set 14) to thetemplate and extension reaction, a sequence in the 5′-end side (13mer)is hybridized with a region starting from 13 bases downstream of the3′-end portion of the primer on a strand extended from the primer;

M13F2L13-12: (SEQ ID NO: 33) TAGCTGTTTCCTGTGTTGTGTGGAATTGTGAGCGGAT;M13R2L13-12: (SEQ ID NO: 34) AGCACATCCCCCTGTGCGGGCCTCTTCGCTATTAC.

Primer set 18: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (24mer: sequenceidentical to that in Primer set 14) to the template and extensionreaction, a sequence in the 5′-end side (13mer) is hybridized with aregion starting from 19 bases downstream of the 3′-end portion of theprimer on a strand extended from the primer, and an antisense primer inwhich after annealing of a sequence placed in the 3′-end side of aprimer (22mer: sequence identical to that in Primer set 14) to thetemplate and extension reaction, a sequence in the 5′-end side (13mer)is hybridized with a region starting from 19 bases downstream of the3′-end portion of the primer on a strand extended from the primer;

M13F2L13-18: (SEQ ID NO: 35) TGGTCATAGCTGTTGTTGTGTGGAATTGTGAGCGGAT;Ml3R2L13-18: (SEQ ID NO: 36) CCTTGCAGCACATGTGCGGGCCTCTTCGCTATTAC.

Primer set 19: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (24mer: sequenceidentical to that in Primer set 14) to the template and extensionreaction, a sequence in the 5′-end side (13mer) is hybridized with aregion starting from 25 bases downstream of the 3′-end portion of theprimer on a strand extended from the primer, and an antisense primer inwhich after annealing of a sequence placed in the 3′-end side of aprimer (22mer: sequence identical to that in Primer set 14) to thetemplate and extension reaction, a sequence in the 5′-end side (13mer)is hybridized with a region starting from 23 bases downstream of the3′-end portion of the primer on a strand extended from the primer;

(SEQ ID NO: 37) M13F2L13: TAATCATGGTCATTGTTGTGTGGAATTGTGAGCGGAT;(SEQ ID NO: 38) M13R3L13: TCGCCTTGCAGCAGTGCGGGCCTCTTCGCTATTAC.

Primer set 20: a combination of a sense primer in which after annealingof a sequence placed in the 3′-end side of a primer (24mer: sequenceidentical to that in Primer set 14) to the template and extensionreaction, a sequence in the 5′-end side (23mer) is hybridized with aregion starting from 25 bases downstream of the 3′-end portion of theprimer on a strand extended from the primer, and an antisense primer inwhich after annealing of a sequence placed in the 3′-end side of aprimer (22mer: sequence identical to that in Primer set 14) to thetemplate and extension reaction, a sequence in the 5′-end side (23mer)is hybridized with a region starting from 23 bases downstream of the3′-end portion of the primer on a strand extended from the primer;

M13F2L23: (SEQ ID NO: 39)CTCGAATTCGTAATCATGGTCATTGTTGTGTGGAATTGTGAGCGGAT; M13R3L23:(SEQ ID NO: 40) CCCAACTTAATCGCCTTGCAGCAGTGCGGGCCTCTTCGCTATTAC.

A reaction mixture (25 μl) of Tris-HCl (20 mM, pH8.8), KCl (10 mM),(NH₄)₂SO₄ (10 mM), MgSO₄ (2 mM), Triton X-100 (0.1%), dNTP (0.4 mM), aprimer pair (100 μmol, resp.), a template DNA (0.05 μg), and Bst DNApolymerase (8U; NEW ENGLAND BioLabs) was prepared, and incubated at 65°C. for 20-120 minutes.

A 5 μl portion of each mixture was subjected to electrophoresis in 3%NuSieve GTG Agarose (manufactured by BMA; purchased from Takara BioInc.; “NuSieve” is the registered trademark of BMA). Results are shownin FIGS. 9 and 10. Samples in respective lanes in these figures areshown in the following Tables 5 and 6.

TABLE 5 Explanation of lanes of electrophoretic photograms in FIG. 9Lane Primer Template Reaction Time (min.) 1 DNA size marker (20 bpladder) 2 Primer set 14 Yes 60 3 Primer set 14 Yes 90 4 Primer set 14Yes 120 5 Primer set 14 No 120 6 Primer set 15 Yes 60 7 Primer set 15Yes 90 8 Primer set 15 Yes 120 9 Primer set 15 No 120 10 Primer set 16Yes 20 11 Primer set 16 Yes 40 12 Primer set 16 Yes 60 13 Primer set 16No 60 14 Primer set 17 Yes 20 15 Primer set 17 Yes 40 16 Primer set 17Yes 60 17 Primer set 17 No 60 18 Primer set 18 Yes 20 19 Primer set 18Yes 40 20 Primer set 18 Yes 60 21 Primer set 18 No 60

TABLE 6 Explanation of lanes of electrophoretic photograms in FIG. 10Lane Primer Template Reaction Time (min.) 1 DNA size marker (20 bpladder) 2 Primer set 19 Yes 20 3 Primer set 19 Yes 40 4 Primer set 19Yes 60 5 Primer set 19 No 60 6 Primer set 20 Yes 20 7 Primer set 20 Yes40 8 Primer set 20 Yes 60 9 Primer set 20 No 60

In Lanes 5, 9, 13, 17 and 21 of respective Figures, no bands other thanthose of stained unreacted primers were observed due to the addition ofno template.

In Lanes 2 and 3 of FIG. 5, amplified products were obtained in areaction time of 90 minutes or more. These products, however, wasamplified product in the form of ladder which was different from theproduct of aimed size. Primer set 14 is used for the amplificationreaction in these lanes. The distance between the region correspondingto the sequence in the 3′-end side of the primer on the extended strandof the primer and the region with which the sequence in the 5′-end sidehybridizes is 50 nucleotides in the sense primers and 53 nucleotides inthe antisense primers. Thus, when the distance between the regioncorresponding to the sequence in the 3′-end side of the primer on theextended strand of the primer and the region with which the sequence inthe 5′-end side hybridizes becomes excessively large as in Primer set 5,it is considered that the aimed amplification products were not obtainedbecause the folding efficiency of the sequence in the 5′-end side ofeach primer is lowered significantly and the synthetic reactionaccording to the invention hardly occurs.

As shown in Lane 7 of FIG. 9, when Primer set 15 was used, it waspossible to obtain the aimed amplification product in a reaction time of90 minutes. Of the low size bands, the band in the vicinity of ca. 240by is an expected product of the synthetic reaction of the invention.

Further, as shown in Lanes 12 and 16 of FIG. 9, and Lanes 4 and 8 ofFIG. 10, it was possible to obtain the aimed amplification product in areaction time of 60 minutes when Primer sets 16, 17, 19 and 20 wereused. Of the low size bands, the band in the vicinity of ca. 240 by isan expected product of the synthetic reaction of the invention.

In addition, as shown in Lane 19 of FIG. 9, it was possible to obtainthe aimed amplification product in a reaction time of 40 minutes or morewhen Primer set 18 was used. Of the low size bands, the band in thevicinity of ca. 240 by is an expected product of the synthetic reactionof the invention.

When the distance between the region corresponding to the sequence inthe 3′-end side of the primer on the extended strand of the primer andthe region with which the sequence in the 5′-end side hybridizes issmall as in Primer set 15, it is considered that a long reaction time isrequired because most of the sequence on a template having the samesequence to which the next primer is to be annealed remains as a doublestrand and the subsequent annealing hardly occurs.

Also, when the distance between the region corresponding to the sequencein the 3′-end side of the primer on the extended strand of the primerand the region with which the sequence in the 5′-end side hybridizes islarge as in Primer sets 6-11, it is considered that a comparatively longreaction time is required because the folding efficiency of the sequencein the 5′-end side of each primer is lowered.

On the other hand, when the distance between the region corresponding tothe sequence in the 3′-end side of the primer on the extended strand ofthe primer and the region with which the sequence in the 5′-end sidehybridizes is not excessively small or excessively large as in Primerset 18, it is considered that the most efficient amplification can beperformed in the invention.

Example 4

Of the amplified products obtained in Examples 1-3, the amplifiedproducts which were believed to have the highest amplificationefficiency were digested with restriction enzymes. A 1 μl portion of thereaction mixture which contained the amplified products obtained withPrimer set 5 described in Example 1 was digested with a restrictionenzyme MboII, a 1 μl portion of the reaction mixture which contained theamplified products obtained with Primer set 12 described in Example 2was digested with a restriction enzyme Bst XI, and a 1 μl portion of thereaction mixture which contained the amplified products obtained withPrimer set 18 described in Example 3 was digested with a restrictionenzyme Pst I. Digestion with restriction enzymes was carried out at atemperature of 37° C. for 3 hours.

Each of the digestion mixtures was subjected to electrophoresis in 3%NuSieve GTG Agarose (manufactured by BMA; purchased from TAKARA BIOINC.; “NuSieve” is the registered trademark of BMA). Results are shownin FIGS. 11, 12 and 13. Sizes of fragments digested with respectiverestriction enzymes which are speculated from the respective basesequences are shown in the side of the electrophoretic photograms. Itwas confirmed from the change of the most part of the undigested bandsinto the bands having speculated sizes after digestion that the aimedamplified products are obtained.

Example 5 Effects of a Variety of Melting Temperature Adjusting Agents

Amplification reaction was conducted with the addition of a variety ofmelting temperature adjusting agents into amplification reactionmixtures in order to examine the effects of the melting temperatureadjusting agents on amplification efficiency. In the same manner as inExample 1, amplification of a human STS DYS237 gene was attempted byusing Human DNA (Clontech) as a template. The primer used was Primer set5 (SEQ ID NO: 9 and SEQ ID NO.: 10) which showed the most preferredamplification efficiency in Example 1.

A reaction mixture (25 μl) of Tris-HCl (20 mM, pH8.8), KCl (10 mM),(NH₄)₂SO₄ (10 mM), MgSO₄ (8 mM), Triton X-100 (0.1%), dNTP (1.4 mM), theprimer pair (1600 nM, resp.), the template DNA, and Bst DNA polymerase(16U; NEW ENGLAND BioLabs) was prepared. The template DNA was added in aconcentration of 100 ng, 10 ng, or 0 ng. To this reaction mixture, 6%DMSO, 0.5 M betaine, 4% formamide, or 10% glycerol as the finalconcentration was added. The mixture was incubated at 60° C. for 90minutes.

After amplification, reaction mixture was subjected to electrophoresisin the same manner as in Example 1. Results are shown in FIG. 14.Samples in respective lanes in FIG. 14 are shown in the following Table7.

TABLE 7 Explanation of lanes of electrophoretic photograms in FIG. 14Melting Temperature Lanes Adjusting Agents Amounts of Template 1 DNAsize marker (20 bp ladder) 2 6% DMSO 100 ng 3 0.5 M Betaine 100 ng 4 4%Formamide 100 ng 5 10% Glycerol 100 ng 6 None 100 ng 7 6% DMSO 10 ng 80.5 M Betaine 10 ng 9 4% Formamide 10 ng 10 10% Glycerol 10 ng 11 None10 ng 12 6% DMSO 1 ng 13 0.5 M Betaine 1 ng 14 4% Formamide 1 ng 15 10%Glycerol 1 ng 16 None 1 ng 17 6% DMSO None 18 0.5 M Betaine None 19 4%Formamide None 20 10% Glycerol None 21 None None 22 DNA size marker (20bp ladder)

In FIG. 14, the band in the vicinity of ca. 160 by is an amplifiedproduct expected by the synthetic reaction of the invention. As apparentfrom FIG. 14, when 100 ng of the template DNA was used, the amplifiedproducts were obtained irrespective of the presence or absence of themelting temperature adjusting agents. On the other hand, when 10 ng ofthe template DNA was used, the amplified products were obtained only inthe presence of the melting temperature adjusting agents. Moreover, when1 ng of the template DNA was used, the bands of the amplified productswere confirmed only in the presence of the melting temperature adjustingagents, and particularly, the most distinct bands of the amplifiedproducts were confirmed in the presence of DMSO (6%) as the meltingtemperature adjusting agent.

It is believed from the above-described results that amplificationefficiency is improved by the addition of the melting temperatureadjusting agents such as DMSO, betaine, formamide or glycerol to thereaction mixture, and particularly, the preferred amplificationefficiency is obtained by the addition of DMSO.

1-17. (canceled)
 18. A primer for synthesizing a nucleic acidcomplementary to a target nucleic acid sequence in a template nucleicacid, comprising in its 3′-end portion a sequence (Ac′) which hybridizesa sequence (A) in the 3′-end portion of the target nucleic acidsequence, and in the 5′-side of said sequence (Ac′) a sequence (B′)which hybridizes the complementary sequence (Bc) of a sequence (B)positioned in the 5′-side of said sequence (A) on the target nucleicacid sequence, wherein in the absence of an intervening sequence betweensaid sequences (Ac′) and (B′), (X−Y)/X is in the range of −1.00 to 1.00,in which X denotes the number of bases in said sequence (Ac′), and Ydenotes the number of bases in the region flanked by said sequences (A)and (B) on the target nucleic acid sequence, and in the presence of anintervening sequence between said sequences (Ac′) and (B′), {X−(Y−Y′)}/Xis in the range of −1.00 to 1.00, in which X and Y have the samemeanings as above, and Y′ denotes the number of bases in saidintervening sequence.
 19. A kit for synthesizing a nucleic acidcomplementary to a target nucleic acid sequence in a template nucleicacid, comprising the primer according to claim
 18. 20. A primer set foramplifying a target nucleic acid sequence in a double-stranded templatenucleic acid, which comprises: (a) a first primer comprising in its3′-end portion a sequence (Ac′) which hybridizes a sequence (A) in the3′-end portion of the target nucleic acid sequence in the first strandof the double-stranded template nucleic acid, and in the 5′-side of saidsequence (Ac′) a sequence (B′) which hybridizes the complementarysequence (Bc) of a sequence (B) positioned in the 5′-side of saidsequence (A) on said target nucleic acid sequence, wherein in theabsence of an intervening sequence between said sequences (Ac′) and(B′), (X−Y)/X is in the range of −1.00 to 1.00, in which X denotes thenumber of bases in said sequence (Ac′), and Y denotes the number ofbases in the region flanked by said sequences (A) and (B) on the targetnucleic acid sequence, and in the presence of an intervening sequencebetween said sequences (Ac′) and (B′), {X−(Y−Y′)}/X is in the range of−1.00 to 1.00, in which X and Y have the same meanings as above, and Y′denotes the number of bases in said intervening sequence; and (b) asecond primer comprising in its 3′-end portion a sequence (Cc′) whichhybridizes a sequence (C) in the 3′-end portion of the target nucleicacid sequence in the second strand of the double-stranded templatenucleic acid, and in the 5′-side of said sequence (Cc′) a sequence (D′)which hybridizes the complementary sequence (Dc) of a sequence (D)positioned in the 5′-side of said sequence (C) on said target nucleicacid sequence, wherein in the absence of an intervening sequence betweensaid sequences (Cc′) and (D′), (X−Y)/X is in the range of −1.00 to 1.00,in which X denotes the number of bases in said sequence (Cc′), and Ydenotes the number of bases in the region flanked by said sequences (C)and (D) on the target nucleic acid sequence, and in the presence of anintervening sequence between said sequences (Cc′) and (D′), {X−(Y−Y′)}/Xis in the range of −1.00 to 1.00, in which X and Y have the samemeanings as above, and Y′ denotes the number of bases in saidintervening sequence.
 21. A kit for amplifying a target nucleic acidsequence in a double-stranded template nucleic acid, comprising theprimer set according to claim 20.